Abstract/Summary

Background: Platelets are crucial in the regulation of haemostasis and thrombosis. Blood fluid dynamics, plasma coagulation factors, vascular endothelial cells (ECs) and the underlying collagen-enriched matrix further contribute to thrombus formation. Studies on the regulation of this process often lack an integrative approach. Aims: In this thesis we aimed to use novel in vitro methods that integrate the various components regulating thrombus formation in human blood to understand: i) the roles of collagens, wall shear-rate and platelet glycoprotein VI (GPVI), ii) the endothelial regulation of platelet and coagulation activation, and iii) the impact of extracellular matrix proteoglycans. Results: Using a microspot-based multiparameter microfluidic approach, we revealed the importance of the protein kinase Syk, operating downstream of GPVI, in thrombus formation induced by different types of collagens expressed in the vascular extracellular matrix. The effects of Syk inhibition on platelet activation under flow were best represented by reduced ‘thrombus signature’ parameters. To recapitulate the in vivo circumstances, a novel vessel-on-a-chip approach was developed, growing partially confluent ECs on collagen- and tissue factor-coated channels. The presence of ECs locally suppressed collagen-induced platelet activation and fibrin formation. The latter being due to ECs thrombomodulin and a glycocalyx enriched in heparan sulphates. At the signalling level, a label-free approach revealed sustained modifications in the platelet phospho-proteome after exposure to ECs, linked to alterations in multiple protein kinases and even extending after platelet stimulation. In addition, we identified a dual platelet-inhibiting and -activating effect by isolated glycocalyx-derived heparan sulphate proteoglycans in thrombus formation. Conclusions: By integrating vascular- and blood-derived components in high-throughput microfluidic methods, this thesis disclosed the presence of potent and complex regulatory mechanisms of ECs and matrix proteoglycans in the control of flow-dependent human thrombus formation. The developed methods promise to be useful for the evaluation of novel antithrombotic agents and for the phenotyping of patients with platelet-related disorders.